Penicillin streptomycin fungizone psf

Urothelial cell cultures were performed as previously described (Kullmann et al., 2009 (link)). Rats were subjected to isoflurane anesthesia, and bladders were resected and placed in cold minimal essential medium (MEM, Invitrogen, Carlsbad, CA, United States) supplemented with HEPES (2.5 g/L, Sigma, St. Louis, MO, United States) and penicillin/streptomycin/fungizone (PSF, 1%, Sigma). The bladder urothelium was incubated in Dispase (2.5 mg/ml, Worthington Biochemical Lakewood, NJ, United States) overnight at 4°C. Urothelial cells were gently scraped, placed in trypsin (0.25% wt/vol, Sigma) for 10–15 min at 37°C, and dissociated by trituration. Cells were suspended in MEM containing 10% FBS and centrifuged at 416 g for 5 min. The cells were suspended in urothelial cell medium (UCM, ScienCell, San Diego, CA, United States) with 1% PSF, centrifuged again, and resuspended in fresh media. Cells were plated on poly-L-lysine-coated glass coverslips and used for Ca²⁺ imaging, 48–96 h after dissociation.

Macrophages were cultured in 75 cm² flasks in Dulbecco’s Modified Eagle’s Medium (DMEM; Lonza, South Africa) containing L-glutamine supplemented with 10% fetal bovine serum (FBS; Gibco, Sigma-Aldrich, Kempton park, South Africa) and 1% penicillin/streptomycin/fungizone (PSF; Sigma-Aldrich, South Africa) at 37 °C with 5% CO₂. Subsequently, cells were seeded in 96-well microtiter plates at 4 × 10⁵ cells/mL and incubated overnight at 37 °C with 5% CO₂ to allow the cells to attach. The cells were consequently activated by the addition of 1 μg/mL of lipopolysaccharide (LPS; Sigma-Aldrich, South Africa), followed by the addition of different concentrations of the extracts. Indomethacin served as a positive control. Cells were incubated for 24 h at 37 °C and 5% CO₂ [25 (link)].